Effects of the Neonicotinoid Insecticide Clothianidin on Southern Leopard Frog (Rana sphenocephala) Tadpole Behavior

  • Jordan N. HoltswarthEmail author
  • Freya E. Rowland
  • Holly J. Puglis
  • Michelle L. Hladik
  • Elisabeth B. Webb


Neonicotinoid insecticides are highly water soluble with relatively long half-lives, which allows them to move into and persist in aquatic ecosystems. However, little is known of the impacts of neonicotinoids on non-target vertebrates, especially at sublethal concentrations. We evaluated the effects of the neonicotinoid clothianidin on the behavior of southern leopard frog tadpoles (Rana sphenocephala) after a 96-h exposure at 6 concentrations, including 0 (control), 0.375, 0.75, 1.5, 3.0, 6.0 µg/L. We quantified total displacement, mean velocity, maximum velocity, and time spent moving of tadpoles for 1 h post-exposure. Total displacement and mean velocity of tadpoles decreased with clothianidin exposure. Maximum velocity decreased linearly with concentration, but there was no relationship between time spent moving and clothianidin concentration. Our results suggest exposure to clothianidin at sublethal concentrations can affect movement behavior of non-target organisms such as tadpoles.


Amphibians Behavior Ecotoxicology Exposure Movement Neonicotinoids 



We would like to thank CERC for allowing us to conduct our experiment in their dilutor system and help with set-up, feeding, and take-down. We also thank S. Michael and J. Patman for allowing us to use BioSense. J. Holtswarth was supported by a University of Missouri College of Agriculture, Food, and Natural Resources Undergraduate Research Scholarship. F. Rowland was supported by a TWA Scholarship. Additional funding was provided by the U.S. Geological Survey Contaminants Biology Program and Toxic Substances and Hydrology Program. Tadpoles were collected and maintained under the University of Missouri Animal Care and Use Committee Protocol 8402 and Missouri Department of Conservation Wildlife Collection Permit 16808. The Missouri Cooperative Fish and Wildlife Research Unit is jointly sponsored by the Missouri Department of Conservation, the University of Missouri, the U.S. Fish and Wildlife Service, the U.S. Geological Survey and the Wildlife Management Institute. Any use of trade, firm or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government.


  1. Albecker MA, McCoy MW (2017) Adaptive responses to salinity stress across multiple life stages in anuran amphibians. Front Zool 14(1):40CrossRefGoogle Scholar
  2. Anderson JC, Dubetz C, Palace VP (2015) Neonicotinoids in the Canadian aquatic environment: a literature review on current use products with a focus on fate, exposure, and biological effects. Sci Total Environ 505:409–422CrossRefGoogle Scholar
  3. Bayci MN (2011) Effects of a neonicotinoid insecticide on larval stages of the green frog, Rana clamitans. Dissertation, Kalamazoo CollegeGoogle Scholar
  4. Bolker B, R Core Development Team (2017) bbmle: tools for general maximum likelihood estimation. R package version 1.0.20Google Scholar
  5. Boone MD, Semlitsch RD (2002) Interactions of an insecticide with competition and pond drying in amphibian communities. Ecol Appl 12(1):307–316CrossRefGoogle Scholar
  6. Calfee RD, Little EE, Puglis HJ et al (2014) Acute sensitivity of white sturgeon (Acipenser transmontanus) and rainbow trout (Oncorhynchus mykiss) to copper, cadmium, or zinc in water-only laboratory exposures. Environ Toxicol Chem 33(10):2259–2272CrossRefGoogle Scholar
  7. Carey C, Bryant CJ (1995) Possible interrelations among environmental toxicants, amphibian development, and decline of amphibian populations. Environ Health Perspect 103(Suppl 4):13–17CrossRefGoogle Scholar
  8. Crosby EB, Bailey JM, Oliveri AN, Levin ED (2015) Neurobehavioral impairments caused by developmental imidacloprid exposure in zebrafish. Neurotoxicol Teratol 49:81–90CrossRefGoogle Scholar
  9. De Lange HJ, Lahr J, Van der Pol JJ et al (2009) Ecological vulnerability in wildlife: an expert judgment and multicriteria analysis tool using ecological traits to assess relative impact of pollutants. Environ Toxicol Chem 28(10):2233–2240CrossRefGoogle Scholar
  10. DeCant J, Barrett M (2010) Clothianidin registration of prosper T400 seed treatment on mustard seed (oilseed and condiment) and Poncho/Votivo seed treatment on cotton. United States Environmental Protection Agency Office of Chemical Safety and Pollution Prevention, Washington, DCGoogle Scholar
  11. Degitz S, Kosian PA, Makynen EA et al (2000) Stage- and species-specific developmental toxicity of all-trans retinoic acid in four native North American ranids and Xenopus laevis. Toxicol Sci 57(2):264–274CrossRefGoogle Scholar
  12. Evelsizer V, Skopec M (2016) Pesticides, including neonicotinoids, in drained wetlands of Iowa’s prairie pothole region. Wetlands 38(2):221–232CrossRefGoogle Scholar
  13. Feng S, Kong Z, Wang X et al (2004) Acute toxicity and genotoxicity of two novel pesticides on amphibian, Rana N. Hallowell. Chemosphere 56(5):457–463CrossRefGoogle Scholar
  14. Finnegan MC, Baxter LR, Maul JD, Hanson ML, Hoekstra PF (2017) Comprehensive characterization of the acute and chronic toxicology of the neonicotinoid insecticide thiamethoxam to a suite of aquatic primary producers, invertebrates and fish. Environ Toxicol Chem 36(10):2838–4288CrossRefGoogle Scholar
  15. Fox J, Weisberg S (2011) An {R} companion to applied regression, 2nd edn. Sage, Thousand OaksGoogle Scholar
  16. Gelman A, Hill J (2006) Data analysis using regression and multilevel/hierarchical models. Cambridge University Press, CambridgeCrossRefGoogle Scholar
  17. Gibbons JW, Winne CT, Scott DE et al (2006) Remarkable amphibian biomass and abundance in an isolated wetland: implications for wetland conservation. Conserv Biol 20(5):1457–1465CrossRefGoogle Scholar
  18. Goodrich B, Gabry J, Ali I, Brilleman S (2018) rstanarm: Bayesian applied regression modeling via Stan. R package version 2.17.4Google Scholar
  19. Gosner K (1960) A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16(3):183–190Google Scholar
  20. Goulson D (2013) An overview of the environmental risks posed by neonicotinoid insecticides. J Appl Ecol 50(4):977–987CrossRefGoogle Scholar
  21. Griffiths RA, Edgar PW, Wong AC (1991) Interspecific competition in tadpoles: growth inhibition and growth retrieval in Natterjack toads, Bufo calamita. J Anim Ecol 60:1065–1076CrossRefGoogle Scholar
  22. Harris ML, Chora L, Bishop CA, Bogart JP (2000) Species- and age-related differences in susceptibility to pesticide exposure for two amphibians, Rana pipiens, and Bufo americanus. Bull Environ Contam Toxicol 64(2):263–270CrossRefGoogle Scholar
  23. Hayasaka D, Korenaga T, Suzuki K et al (2012) Cumulative ecological impacts of two successive annual treatments of imidacloprid and fipronil on aquatic communities of paddy mesocosms. Ecotoxicol Environ Saf 80:355–362CrossRefGoogle Scholar
  24. Hecnar SJ (1995) Acute and chronic toxicity of ammonium nitrate fertilizer to amphibians from southern Ontario. Environ Toxicol Chem Int J 14(12):2131–2137CrossRefGoogle Scholar
  25. Hladik ML, Calhoun DL (2012) Analysis of the herbicide diuron, three diuron degradates, and six neonicotinoid insecticides in water-method details and application to two Georgia streams. Sci Investig Rep 2012 2012:5206Google Scholar
  26. Hladik ML, Kolpin DW, Kuivila KM (2014) Widespread occurrence of neonicotinoid insecticides in streams in a high corn and soybean producing region, USA. Environ Pollut 193:189–196CrossRefGoogle Scholar
  27. Köhler H-R, Triebskorn R (2013) Wildlife ecotoxicology of pesticides: can we track effects to the population level and beyond? Science 341(6147):759–765CrossRefGoogle Scholar
  28. Kuechle KJ, Webb EB, Mengel D, Main AR (2019) Factors influencing neonicotinoid insecticide concentrations in floodplain wetland sediments across Missouri.  Environ Sci Technol.
  29. Leary SL, Underwood W, Anthony R et al (2013) AVMA guidelines for the euthanasia of animals, 2013th edn. American Veterinary Medical Association, SchaumburgGoogle Scholar
  30. Lee-Jenkins SS, Robinson SA (2018) Effects of neonicotinoids on putative escape behavior of juvenile wood frogs (Lithobates sylvaticus) chronically exposed as tadpoles. Environ Toxicol Chem 37(12):3115–3123CrossRefGoogle Scholar
  31. Lewis KA, Tzilivakis J, Warner D, Green A (2016) An international database for pesticide risk assessments and management. Hum Ecol Risk Assess Int J 22(4):1050–1064. CrossRefGoogle Scholar
  32. Main AR, Headley JV, Peru KM et al (2014) Widespread use and frequent detection of neonicotinoid insecticides in wetlands of Canada’s Prairie Pothole Region. PLoS ONE 9(3):e92821CrossRefGoogle Scholar
  33. Matsuda K, Buckingham SD, Kleier D et al (2001) Neonicotinoids: insecticides acting on insect nicotinic acetylcholine receptors. Trends Pharmacol Sci 22(11):573–580CrossRefGoogle Scholar
  34. Miles JC, Hua J, Sepulveda MS et al (2017) Effects of clothianidin on aquatic communities: evaluating the impacts of lethal and sublethal exposure to neonicotinoids. PLoS ONE 12(3):e0174171CrossRefGoogle Scholar
  35. Moe TA (2017) Sub-lethal and lethal effects of a neonicotinoid pesticide on the development of Northern leopard frog tadpoles. All NMU Master’s Theses, p 153. Accessed 25 Oct 2017
  36. Morrissey CA, Mineau P, Devries JH et al (2015) Neonicotinoid contamination of global surface waters and associated risk to aquatic invertebrates: a review. Environ Int 74:291–303CrossRefGoogle Scholar
  37. Patman J, Michael S, Lutnesky MMF, Palaniappan K (2018) BioSense: real-time object tracking for animal movement and behavior research. In: IEEE applied imagery pattern recognition workshop (AIPR), 2018Google Scholar
  38. R Core Team (2018) R: a language and environment for statistical computing. R Foundation for Statistical Computing, ViennaGoogle Scholar
  39. Robinson SA, Richardson SD, Dalton RL et al (2017) Sublethal effects on wood frogs chronically exposed to environmentally relevant concentrations of two neonicotinoid insecticides. Environ Toxicol Chem 36(4):1101–1109CrossRefGoogle Scholar
  40. Sánchez-Bayo F (2012) Insecticides mode of action in relation to their toxicity to non-target organisms. Environ Anal Toxicol S4:002. Google Scholar
  41. Sánchez-Bayo F, Goka K, Hayasaka D (2016) Contamination of the aquatic environment with neonicotinoids and its implication for ecosystems. Front Environ Sci 4:71CrossRefGoogle Scholar
  42. Schmidt K, Blanchette ML, Pearson RG et al (2017) Trophic roles of tadpoles in tropical Australian streams. Freshw Biol 62(11):1929–1941Google Scholar
  43. Simon-Delso N, Amaral-Rogers V, Belzunces LP, Bonmatin JM, Chagnon M, Downs C, Furlan L, Gibbons DW, Giorio C, Girolami V, Goulson D (2015) Systemic insecticides (neonicotinoids and fipronil): trends, uses, mode of action and metabolites. Environ Sci Pollut Res 22(1):5–34CrossRefGoogle Scholar
  44. Stan Development Team (2018) RStan: the R interface to Stan. R package version 2.18.2Google Scholar
  45. Swanson JE (2017) Amphibian occupancy and effects of habitat use on pesticide exposure in Iowa agricultural wetlands. Dissertation, Iowa State UniversityGoogle Scholar
  46. Van Meter RJ, Glinski DA, Hong T et al (2014) Estimating terrestrial amphibian pesticide body burden through dermal exposure. Environ Pollut 193:262–268CrossRefGoogle Scholar
  47. Wilbur HM (1980) Complex life cycles. Annu Rev Ecol Syst 11(1):67–93CrossRefGoogle Scholar
  48. Yao Y, Vehtari A, Simpson D, Gelman A (2018) Using stacking to average Bayesian predictive distributions. Bayesian Anal. Google Scholar

Copyright information

© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2019

Authors and Affiliations

  • Jordan N. Holtswarth
    • 1
    • 6
    Email author
  • Freya E. Rowland
    • 2
    • 7
  • Holly J. Puglis
    • 3
  • Michelle L. Hladik
    • 4
  • Elisabeth B. Webb
    • 1
    • 5
  1. 1.School of Natural ResourcesUniversity of MissouriColumbiaUSA
  2. 2.Division of Biological SciencesUniversity of MissouriColumbiaUSA
  3. 3.Columbia Environmental Research CenterU.S. Geological SurveyColumbiaUSA
  4. 4.California Water Science CenterU.S. Geological SurveySacramentoUSA
  5. 5.U.S. Geological SurveyMissouri Cooperative Fish and Wildlife Research UnitColumbiaUSA
  6. 6.Department of Natural Resources and Environmental SciencesUniversity of IllinoisUrbanaUSA
  7. 7.School of Forestry and Environmental StudiesYale UniversityNew HavenUSA

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